The hematopoietic stem cell (HSC) through proliferation and differentiation gives rise to most, if not all, of the cells in the hematopoietic system. Thus, HSCs are ideal candidates for disease therapy and are attractive target cells for delivery of genes and gene products to a host animal. However, it is often difficult to isolate sufficient HSCs from tissue such as bone marrow for a number of reasons. The number of HSCs in the tissue can be low compared to non-HSCs and the correct identification of HSCs capable of repopulating the bone marrow of a host animal has been difficult and inconsistent. Difficulties in ex vivo expansion of hematopoietic stem cells (HSCs) have greatly hampered their clinical utility as well as studies of their biological properties (J. Domen, I. L. Weissman, Mol Med Today 5, 201-8 (May, 1999)). Although numerous attempts have been made to increase the number of long-term HSCs (LT-HSCs) in culture, there has only been limited success. The use of stromal cell lines and combinations of cytokines have, at best, lead to maintenance or modest expansion of murine long-term (LT)-HSC activity (K. A. Moore, H. Ema, I. R. Lemischka, Blood 89, 4337-47 (Jun. 15, 1997); C. C. Fraser, C. J. Eaves, S. J. Szilvassy, R. K. Humphries, Blood 76, 1071-6 (Sep. 15, 1990); and C. L. Miller, C. J. Eaves, Proc Natl Acad Sci USA 94, 13648-53 (Dec. 9, 1997)). The introduction of exogenous transcription factors can significantly expand HSCs (K. D. Bunting, J. Galipeau, D. Topham, E. Benaim, B. P. Sorrentino, Ann NY Acad Sci 872, 125-40; discussion 140-1 (Apr. 30, 1999); B. Varnum-Finney et al., Nat Med 6, 1278-81 (November 2000); J. Antonchuk, G. Sauvageau, R. K. Humphries, Cell 109, 39-45 (Apr. 5, 2002); and T. Reya et al., Nature 423, 409-14 (May 22, 2003)), but this approach may have undesirable outcomes for recipients. Furthermore, it appears that in vivo HSC surface phenotypes do not correlate to expansion of HSC activity (K. D. Bunting, J. Galipeau, D. Topham, E. Benaim, B. P. Sorrentino, Ann NY Acad Sci 872, 125-40; discussion 140-1 (Apr. 30, 1999)). Therefore, the in vivo stem cell phenotype does not necessarily predict the hematopoietic repopulating potential of the ex vivo cultured cells.
There is a need, therefore, for improved methods of ex vivo cell culture systems capable of expanding hematopoietic cells that maintain pluripotency. Furthermore, there is a need for methods that allow the identification of ex vivo expanded cells that retain such pluripotency.